Tuberculosis (TB) is one of the world's greatest health problems, causing approximately 3 million deaths per year. Despite continuing increases in global morbidity and mortality, therapeutic and preventative options for TB remain severely limited. The central feature of TB pathogenesis is infection and intracellular survival of Mycobacterium tuberculosis (Mtb) within human macrophages. Following phagocytosis, Mtb evades the normally potent antimicrobial defenses of innate immunity by inhibiting the maturation of its phagosome to a microbicidal phagolysosome. The molecular mechanisms by which Mtb blocks phagosomal maturation and survives intracellularly are incompletely understood. The long-term goal of this project is to define the molecular mechanisms of tuberculous pathogenesis, to provide a foundation for improved therapies and vaccines. Recently, we demonstrated that live, virulent Mtb, but not killed Mtb, inhibit macrophage Ca2+-signaling, and that this defect in host activation directly contributes to inhibition of phagosomal maturation and promotion of the bacilli's intracellular survival. Important gaps in our knowledge include: (1) the mycobacterial determinants responsible for inhibition of macrophage Ca2+-signaling, and (2) the macrophage targets of Mtb-induced inhibition during this critical phase of the host-pathogen interaction. The hypotheses are: (a) sphingosine kinase (SK) is a critical target of macrophage deactivation by live Mtb, and (b) inhibition of SK is causally related to defective Ca2+-signaling, inhibition of phagosome maturation, and the survival of Mtb within human macrophages. We will investigate these hypotheses by pursuing the following Specific Aims: (1) Characterize the activation of macrophage SK during phagocytosis of killed Mtb and its role in Ca2+-signal transduction and phagosome maturation. (2) Determine whether inhibition of SK-mediatedCa2+-signaling by live Mtb is causally related to defective phagosome maturation and intracellular viability. In Aims 1 and 2, pharmacological, biochemical, and genetic approaches will be used to modulate specific signaling pathways. (3) Determine the component(s) of Mtb responsible for inhibition of macrophage SK- and Ca2+-mediated activation. A genetic approach of screening a transposon mutant library of Mtb and a biochemical approach of direct assessment of subcellular fractions of Mtb for effects on macrophage SK-andCa2+-mediated signal transduction will be undertaken. [unreadable] [unreadable]